1) Development of functional neuro-vascular networks. We have developed a novel in vivo Ca2+ imaging system of embryonic forelimb skin using sensory neuron-specific Ca2+ indicator mice: a knock-in mouse line in which the expression of GCaMP3, a calcium indicator whose green fluorescent intensity is driven by a sensory neuron-specific phosphoinositide-binding protein (Pirt) promoter. The limb skin is dissected from Pirt-GCaMP3 embryos, and sensory neurons are stimulated by capsaicin. We have successfully demonstrated GCaMP3 fluorescence in response to the capsaicin stimuli. We are currently attempting 1) to carry out the time course analysis to study the relationship between sensory activity and the processes of neurovascular co-patterning and 2) to examine what happens to sensory activity in the mutants having neuro-vascular mis-patterning. 2) Neuro-vascular networks in pathological situations including obesity-related nerve disorders. We have developed a high-resolution whole-mount imaging method to analyze neuro-vascular branching in the entire ear skin of adult mice. This method enabled us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels in the animal models of obesity and type 2 diabetes, with comprehensive quantification measurements (Yamazaki et al. 2018 Sci Rep). We are currently investigating the functional consequence of defective neuro-vascular branching using an in vivo Ca2+ imaging system of adult ear skin using sensory neuron-specific Pirt-GCaMP3 mice. 3) Contribution of non-vascular cells in organ-specific vascular development. We are interested in examining how the multiple vascular cell types (endothelial cells, pericytes, and VSMCs) assemble to form an organ-specific vascular network. Our recent studies have demonstrated that there are tissue-localized myeloid progenitor-derived pericytes in the vasculature of the ectoderm-derived tissues such as skin and brain. Further genetic studies revealed that TGF- influences differentiation of myeloid progenitors into pericytes (Yamazaki et al. 2017 Cell Rep). To define pericyte heterogeneity at the molecular level, we are currently carrying out a single cell RNA-seq analysis of freshly FACS-isolated PDGFR+ pericytes from embryonic skin, in combination with a total RNA-seq analysis of and myeloid cell-derived and mesenchymal cell-derived pericytes.